The present application is a continuing application based on U.S. Provisional Application Ser. No. 60/043,545, filed Apr. 14, 1997, the entire contents of which is specifically incorporated herein by reference in its entirety.
1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising native, site-specifically mutagenized, and synthetic peptides comprising portions of the human estrogen receptor, or estrogen receptor co-activator proteins. The invention further provides nucleic acid compositions encoding these peptide and protein compositions. Also provided are methods for synthesizing phosphotyrosyl and malonyltyrosyl peptide derivatives of these peptides and their use as antiestrogen compositions in the treatment of breast cancers, the preparation of pharmaceutical compositions, diagnostic kits, and the development of related assays for use in antitumor therapies.
1.2 Description of the Related Art
1.2.1 Breast Cancer
Breast cancer is the most common form of cancer among women, affecting about one in eight women. Approximately 185,700 new cases are diagnosed in the U.S. annually, and breast cancer is responsible for about 44,560 deaths in the U.S. per year. While predominantly observed in women, 1,400 cases of breast cancer are diagnosed annually in men, and 260 men die of breast cancer per year. Breast cancer first manifests itself as a painless lump, detectable by self-examination and clinical breast exams including mammograms. Commonly, growth initiates in the lining of the ducts or in the lobules of the breast. Current clinical treatments include mastectomy (removal of the entire breast) or lumpectomy (removal of the tumor and surrounding tissue) for localized tumors. Chemotherapy, radiotherapy, or hormone-blocking therapy may be further used to control cancerous cells. Breast cancer cells can metastasize to the lymph nodes, skin, lungs, liver, brain, or bones. Metastasis may occur early or late in the disease progression, although typically metastasis occurs once the cancerous growth reaches a size of about 20 mm. Metastasis is achieved by cells breaking away from the parental mass and entering either the bloodstream or the lymphatic system.
Genetic inheritance appears to play a role in about 5-10% of breast cancer patients. Mutations in the BRCA1, BRCA2, and p53 tumor suppressor genes have been observed to confer high risks of breast and ovarian cancers. BRCAI mutations are present at between 1 in 300 to 1 in 800 females. In the BRCA1 gene, over 200 different mutations have been discovered to date. The mutations observed are not localized to a single region, further complicating genetic analysis. Greater than 80% of the observed mutations result in a truncated form of the BRCA1 protein. Individuals with familial hereditary BRCA1 possess one normal and one mutant form of the gene, and are therefore much more likely to develop breast cancer. It is estimated that women with a hereditary BRCA1 mutation are about 76% likely to develop breast cancer by 70 years of age.
BRCA2 has been identified on chromosome 13q through linkage analysis of 15 breast cancer families that did not demonstrate BRCAI linked breast cancer. Unlike BRCA1 mutations, BRCA2 does not substantially elevate the risk of ovarian cancers. The BRCA2 gene encodes a protein of 3,418 amino acids, many of which are acidic or basic. Most mutations observed involve base deletions that alter the reading frame, and result in a premature truncation of the protein. BRCA1 and BRCA2 account for about 45% of familial inherited breast cancers each, leaving 10% for one or more additional genes. Interestingly, all male breast cancers appear to be due to mutations in the BRCA2 gene. Mutations found in breast tumor p53 genes are commonly single base pair changes which result in variants with increased cellular half lives. Altered p53 proteins have been observed in 20-25% of breast cancers.
1.2.2 Steroid Hormone Receptors
The steroid/thyroid hormone receptors are ligand-dependent transcription factors that function by binding to hormone response elements on target genes and regulating transcription (Evans, 1988). Although receptor-associated coactivators have been identified, the processes controlling steroid-specific gene transcription are poorly understood (LeDouarin et al., 1995). Most steroid/thyroid hormone receptors, including the human estrogen receptor (hER), bind to their hormone response elements as hetero- or homodimers (Kumar and Chambon, 1988; Kliewer et al., 1992), and it has been suggested that the dimerization of the steroid/thyroid hormone receptors is mediated, in part, through a leucine zipper motif in the carboxyl termini of the receptors (Forman et al., 1989; Fawell et al., 1990).
Antiestrogen therapy has had a significant impact on survival in patients with breast cancer (Jaiyesimi et al., 1995). The presence of estrogen receptor in breast tumors identifies those patients with a lower risk for disease recurrence and a better response to endocrine intervention. However, as breast cancer progresses, it usually becomes resistant to estrogens, and most patients no longer respond to treatment with tamoxifen or other antiestrogens. Results of new studies suggest that disruption of phosphotyrosine-dependent pathways may offer an alternate approach to antiestrogen treatment (Reddy et al., 1992). Modulation of the biologic activity of ER by estrogen and by tyrosine kinase signaling pathways appears to be functionally related to phosphorylation of specific conserved tyrosine residues in ER (Migliaccio et al., 1989; Castoria et al., 1993; Arnold et al., 1995; Pietras et al., 1995).
The ER is a phosphoprotein found in more than two-thirds of human breast tumors (Arnold and Notides, 1995; Weis et al., 1996; White et al., 1997). Estrogen binding to ER is thought to induce conformational changes in the receptor leading to formation of homodimers and association of the hormone-ER complexes with defmed palindromic DNA sequences termed estrogen responsive elements. EREs are usually located upstream of estrogen-responsive genes and act to regulate gene transcription and cell growth (Green and Chambon, 1988; Kato et al., 1995). Transcription is induced by two separate activation functions of the ER, an amino-terminal AF-1 region and a carboxy-terminal AF-2 region located in the hormone-binding domain of ER.
Phosphorylation of tyrosine in ER may be central to the regulation of receptor dimerization and the subsequent interaction with ERE in DNA (Castoria et al., 1993; Arnold et al., 1995; Pietras et al., 1995; Arnold and Notides, 1995; Arnold et al., 1995). New data suggest that Tyr537 may be required to maintain ER in a transcriptionally inactive state. Inactive ER is a monomer and upon estrogen-induced phosphorylation at Tyr537 and serine residues, it forms an active dimer that can bind ERE (Arnold et al., 1995; Pietras et al., 1995; Arnold and Notides, 1995; Arnold et al., 1995). Phosphotyrosine and neighboring amino acid residues on one ER monomer may provide a specific binding site for association with complementary domains on other ER monomers.
While the dimerization of most steroid hormone receptors is required for binding to DNA, accessory proteins and post-translational phosphorylation have also been implicated in DNA binding (Onate et al., 1994; Shuai et al., 1994; Hou et al., 1994). Arnold et al., (1995) demonstrated the phosphorylation of steroid/thyroid hormone receptors modulated their DNA binding affinity. The phosphorylation of the retinoic acid and progesterone receptors increases, while the phosphorylationof thyroid hormone receptor-.alpha..sub.2 and nerve growth factor-I-B decreases their affinity for their respective response elements (Rochette-Egly et al., 1995; Denner et al., 1989; Katz et al., 1995; Hirataetal, 1993).
Human ER (hER), like other members of the steroid/thyroid hormone receptor superfamily, undergoes a hyperphosphorylation at serine residues following hormone binding (Denton et al., 1992). The dephosphorylation of hER with potato acid phosphatase reduces but does not eliminate the receptor's affinity for an ERE (Denton et al., 1992). Arnold et al., (1994) have shown that casein kinase II specifically phosphorylates hER at serine 167.
An estradiol-independent phosphorylation site at Tyr537 in the carboxyl-terminus of the hER has been identified by amino acid sequencing of .sup.32 P-labeled tryptic peptides of the hER (Arnold et al., 1995). Furthermore, the Src family tyrosine kinases, p60.sup.c-src and p56.sup.lck, were shown to specifically phosphorylate Tyr537 on the hER, while protein-tyrosine phosphatase 1 (SHPTP1) dephosphorylated phosphotyrosine-537 (Arnold et al., 1995). Interestingly, the tyrosine kinase activity of p60.sup.c-src in human breast cancers has been shown to be elevated as compared with other cancers (Jacobs and Rubsaamen, 1983). The human MCF-7 mammary carcinoma cell line overexpresses p.sub.60.sup.c-src and has been a useful paradigm for investigating estrogen-dependent processes associated with human breast cancers (Katzenellenbogen et al., 1987).
Arnold et al., (1995) demonstrated that the phosphorylation of Tyr537 is a regulatory mechanism that controls the capacity of the hER to undergo the monomer to dimer transition, and that the phosphorylation of Tyr537 is a prerequisite for the estrogen-dependent hyperphosphorylation of the serine residue(s), nuclear retention, and DNA binding of the hER.
1.2.3 Estrogens and the Proliferation of Breast Cancer
Estrogens and peptide growth factors control the proliferation of breast cells. Alterations in the receptors for these agonists occur in human cancers in nature and lead to disruption of growth regulation (Harris et al., 1992). Among growth factor receptors, the most frequently implicated in human cancers have been members of the class I receptor tyrosine kinase family (erbB). ErbB tyrosine kinase receptors are overexpressed in two-thirds of human breast cancers and are associated with malignant transformation (Slamon et al., 1987; Slamon et al., 1989a; 1989b; Harris et al., 1992; Dougall et al., 1994). These receptors include the HER-2 (erbB2) and HER-3 (erbB3) proteins which, together, constitute a high affinity functional receptor for heregulin (HRG), a ligand implicated in the autocrine/paracrine growth of breast epithelial cells (Carraway and Cantley, 1994; Sliwkowski et al., 1994). Receptors for estrogen are part of a family of steroid hormone receptors related to the viral erbA gene (Green and Chambon, 1988), and like the erbB proteins these receptors may play important pathogenic roles in breast cancer. Cross-oupling between erbB and estrogen receptor (ER) signal pathways in rodent uterine tissues has been reported (Ignar-Trowbridge et al., 1992) and is reminiscent of the cooperativity between viral erbA and erbB oncogenes in the malignant transformation of avian hematopoietic cells (Beug and Graf, 1989). Direct interaction between erbB signal pathways and ER in human breast cancer cells is the subject of several current studies (Pietras et al., 1995).
Upon estradiol binding, ER interacts with specific estrogen-response elements (ERE) in the vicinity of target genes and modulates their transcription (Green and Chambon, 1988; Smith et al., 1993). The HER-2 receptor, with intrinsic tyrosine kinase activity, is believed to promote signal transduction along specific phosphorylation cascades (Harris et al., 1992; Silvennoinenet al., 1993; Dougall et al., 1994), with recruitment of proteins that serve as a link in activation of ras, inositol triphosphate, and, possibly, other signaling pathways to the nucleus (Silvennoinen et al, 1993). Phosphorylation of ER on tyrosine and/or serine residues has been associated with functional changes in both hormone binding and nuclear localization (Arnold et al., 1994; Kuiper et al., 1994; Le Goff et al., 1994) and may be a link to kinase-mediated growth factor pathways. Blockade of estrogen-induced growth of breast tumor cells by tyrosine kinase inhibitors provides further evidence of the importance of tyrosine kinase pathways in estrogen action (Reddy et al., 1992).
Expression of either HER-2 or ER in human breast cancer provides important prognostic information (Slamon et al., 1987; Slamon et al., 1989b; Nicholson et al., 1990; Benz et al., 1993; Wright et al., 1992; Borg et al., 1994; Elledge et al., 1994). There are considerable data showing an association between HER-2 overexpression and the ER-negative phenotype (Zeillinger et al., 1989; Adnane et al., 1989), and failure of antiestrogen therapy in patients with breast cancer correlates with erb B receptor expression (Nicholson et al., 1990; Wright et al., 1992). In view of the above data, a greater understanding of the possible influence of erb B genes on the estrogen response is needed. Although ER is known to modulate HER-2 gene expression (Read et al., 1990; Russell et al., 1992), the inventor postulates that reciprocal regulation of ER by ErbB pathways may also occur, fostering hormone-independent growth in breast cancer.